1. Field of the Invention
The present invention is concerned with methods for ocular administration of drugs and drug preparations for this purpose. More particularly, the invention is concerned with such methods and preparations including one or more selected drugs and a peptide serving to enhance the transport of the drug(s) across ocular tissues. The preparations may also include other ingredients such as a buffer and an acrylic polymer.
2. Description of the Prior Art
Ocular conditions such as glaucoma, ocular inflammations and infections often require ocular administration of drugs for most efficient treatment. This stems from the poor accessibility of a number of ocular regions to systemic circulation and the resultant fact that the administered drugs are poorly retained in the ocular regions. Unfortunately, drug transport across the cornea is inefficient, owing to the fact that the intrinsic conjunctional epithehlia forms tight junctions with high resistance to ocular delivery. Moreover tear washout and the blinking reflex dilutes and washes out the administered drugs.
These factors contribute to intra-ocular bioavailability of generally less than 10% for drugs. Consequently, this low efficiency necessitates the use of high concentrations of the active ingredients in the ophthalmic formulations. Furthermore, the ocular barrier tissues are highly sensitive to high local concentrations of drugs or vehicles. Generally, lipophilic molecules show the best intrinsic permeation rates across the cornea. However, many therapeutically important compounds such as antibiotics are more polar and hydrophilic molecules, and are thus poorly transported across the corneal epithelium.
The treatment of bacterial keratitis, an ocular infection of the corneal stroma, exemplifies the problems associated with ocular administration of drugs. This condition is reported in approximately 30,000 cases annually in the United States (including bacteria, fungus, and acanthamceba). Gram-negative Pseudomonas species and gram-positive Staphylococci and Streptococci strains are the most frequently identified pathogenic organisms. While less severe forms of bacterial keratitis may not lead to corneal scarring and sight loss, most cases are at risk for subsequent loss of vision. Untreated corneal infections may result in corneal perforation, with the potential for development of endophthalmitis and loss of the eye. Rapid diagnosis and timely therapeutic intervention are essential because ocular destruction can occur rapidly, within 24 hours with highly virulent organisms. The appearance of antibiotic resistant bacterial strains requires an increasing use of more exotic antimicrobials, many of which are highly polar in character and therefore poorly transported across epithelia.
Many ophthalmic medications are formulated for instillation as eye drops. The obvious accessibility of the front of the eye belies the difficulty of efficiently delivering certain classes of drugs across the outer epithelial layer. Further considerations are the small volume (30 μL) that can be delivered to the eye and rapid clearance observed in these tissues. Low bioavailability of drugs from eye drops is mainly due to tear production, low absorption, low residence time, and the impermeability of corneal epithelium. These factors severely limit the effectiveness of this route of administration. Various formulations have been developed to increase and maintain drug concentrations prior to transport. One approach has been the addition of viscose polymers such as the poly (acrylic acid) derivatives Carbopol 1342P NF, Carbopol 974P and Carbopol 980 NF. These additives increase contact time with the eye. Another emerging approach is the use of therapeutic contact lenses wherein several different hydrogel materials are presoaked in different ophthalmic drugs. These lenses are then tested for drug uptake and delivery.
The barrier function of epithelial cells is performed by tight junctions in the form of complex, highly regulated, protein structures. Transient openings of these junctions are required for a variety of bodily functions including sperm maturation, extravasation of lymphocytes across endothelia and nutrient uptake associated with activity of the Na+/glucose transporter. The cytoskeleton of actin microfilaments, associated with myosin and other cellular proteins, maintains the morphology of epithelial cells. An intracellular ring of actin and myosin at the apical/lateral interface (the perijunctional actomyosin ring) provides a scaffold for the tight junctions between epithelial or endothelial cells. The primary transmembrane structural components of tight junctions are the claudin family proteins, junctionall adhesion molecules (JAM) and occluden. These proteins interact directly with the ZO family proteins, which link them to the perijunctional ring of the cytoskeleton. These proteins interact with several regulatory/signaling molecules. The ZO proteins contain a guanylate kinase (GUK) domain as well as a src homology 3 (SH3) domain and a PDZ domain. The atypical PKC isotype specific interacting protein (ASIP) and the ras binding protein AF-6 also contain PDZ domains, and have been shown to associate with junctional complexes. PKC phosphorylates occluden, which results in its translocation to the tight junction. In subconfluent epithelial cell cultures, ZO-1 localizes to the nucleus but is located at the junctions in confluent cultures of epithelial cells. Myosin light chain kincase phosphorylation of the myosin II (regulatory subunit) is associated with contraction of the perijunctional ring and increases in paracellular permeability. Protein kinase A (PKA) activation increases conductance, but not permeability to large molecules across tight junctions, while activation of PKC increases paracellular permeability. Barrier function of the tight junction is also affected by calcium levels, which maybe under the control of PKC. Rho GTPase family members control organization of the actin cytoskeleton, (specifically cdc42). Rab GTPase proteins, which play a regulatory role in vesicular trafficking, such as rab13 and rab3b, appear to play a role in junctional regulation that remains undefined. These observations demonstrate that numerous cellular components might be targeted to modulate the paracellular conductance.
Endothelial tight junctions also share many components with epithelial tight junctions although distinct extracellular modulators impinge on their function. A variety of techniques are currently being investigated to selectively and transiently circumvent the barrier function of epithelia and endothelia. For example, pharmaceuticals are being linked to actively transported peptides as a means to cross the blood-brain barrier. While this allows very selective targeting, the method requires a unique synthetic process for every transported compound. Alternatively, methods are being developed to reduce epithelial tight junctions enough to allow large molecules to diffuse to the interstitial space. Both calcium chelators and surfactants have been employed, but have unacceptable side effects including global changes in cell function and diminished cell adhesion. Alternatively, the zonula occludens toxin of Vibrio cholerae (ZOT) provides a naturally occurring alternative for increasing the permeability of small intestine epithelia. ZOT and its eukaryotic homologue, zonulin, interact with an epithelial membrane receptor that leads to a reduction in epithelial resistance, presumably by activation of PKCa. The effects of ZOT are rapid in onset (<20 minutes) and readily reversible upon washout. Thus ZOT is an excellent candidate as an adjunct to standard therapy to increase oral bioavailability of large molecules. In fact, ZOT has been used to increase the permeation of anticonvulsant drugs across epithelial monolayers, to increase the uptake of PEG 4000 from rabbit small intestine and into the bloodstream, and to reversibly increase intestinal permeability to insulin and immunoglobulins. In diabetic rats, the bioavailability of oral insulin was sufficient to control blood glucose to the same degree as parenteral administration. However, ZOT has some drawbacks as a more generalized therapeutic in that it is a large peptide (399 a.a.) and has a relatively small therapeutic target. Effects are observed only in the small intestine where distinct receptors are present. It was recently reported that an 8 a.a. peptide could fully inhibit the effects of ZOT on small intestine.
Synthetic peptides based upon portions of naturally occurring proteins have been employed as epithelial ion channels for a number of years. In addition to developing potential therapeutics for cystic fibrosis, these synthetic channel-forming peptides have greatly expanded the understanding of channel structure. The M2 segments of both the acetylcholine receptor delta subunit and the glycine receptor alpha subunit have been employed. In each case, ion selective channels were produced. Amphipathic transmembrane α-helices are thought to be the structural motif responsible for formation of the aqueous-facing pore region of ion channels. The M2GlyR peptide has single-channel conductances of 25 pS and 49 pS with open lifetimes in the millisecond time. These observations are in good agreement with single-channel properties described for glycine receptor conductance measured in inside-out patches from cultured rat spinal cord neurons. Lysine residues have been added to either terminus of the M2GlyR peptide (NK4-M2GlyR, CK4-M2GlyR) to increase their aqueous solubility. Two-dimensional total coherence spectroscopy (TOCSY-NMR) and reverse phase HPLC studies revealed that peptides became less aggregated in aqueous solution as the number of positively charged amino acids at the termini was increased. The ability of CK4-M2GlyR and other M2GlyR peptides to induce apical chloride secretion from monolayers of Manin-Darby canine kidney cells (MDCK) was determined by measuring peptide-induced increases in ion transport. NK4-M2GlyR and CK4-M2GlyR applied to the apical membrane of human epithelial cell monolayers induced transepithelial Cl− and fluid secretion, although with differing potency.
The eye has been described as an immune privileged site where immunity is suppressed. Comeal allograft transplants appear tolerated rather than rejected like solid organ transplants such as heart, lung, pancreas, bowel, kidney and skin. This immune suppression is hypothesized to occur as a result of the absence of both vascular and lymphatic systems. There is also an anterior chamber-associated immune deviation (ACAID) that is characterized by a suppression of some forms of systemic immunity after the administration of antigen into the eye. A number of reports have shown that cytokines such as IL-2, IL-4, IL-12, or granulocyte macrophage-colony stimulating factor secreted from neural derived tumors generate anti tumor immunity.
A literature survey reveals that there are no references dealing specifically with corneal immuno/inflammatory responses upon topical application of synthetic peptides. There are studies that describe experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU) induced in lab animals with the systemic exposure of different peptides sequences derived from myelin basic protein (MBP), HLA-B, interphotoreceptor retinoid-binding protein (IRBP), and fragments from the human S-Antigen (S-Ag).
While the eye's reduced immunological potential appears to make this tissue a candidate for peptide-based therapeutics, full-blown immune responses are known. Responses have been seen in connection with corneal allografts and herpes simplex infections. Herpetic corneal stromal keratitis (HSK) is a T cell-controlled, immunoinflammatory lesion resulting from Herpes simplex virus infection. Release of proinflammatory molecules such as IL-1 has been implicated in HSK pathogenesis. Recent results suggest that early treatment with IL-1 receptor antagonist (IL-1 ra) protein reduces the severity of HSK. Other bacterial infections, such as those caused by Staphylococcus aureus and Pseudomonas aeruginosa can also lead to immuno/inflammatory responses.
When an immune response is triggered a number of cellular events mediate the corneal immuno/inflammatory response. These include activation and migration of local antigen-presenting cells (APCs), including Langerhans cells (LCs), up-regulation in pleiotropic proinflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) that can mediate a wide array of immune functions in addition to up-regulating protease expression. Additionally, chemokines attract both non antigen-specific inflammatory cells such as neutrophils and CD4+ T helper type 1 (Th1) cells that mediate most of the destruction in the cornea. It has been shown that the cornea is a potent producer of nitric oxide (NO) a molecule that is toxic to various pathogens and possesses immunomodulatory properties. The production of various cytokines, including interleukin (IL)-1, IL-6, IL-8, IL-18, interferon (IFN)-γ and TGF-β, has also been demonstrated in cultured corneal cells in vitro. Both dependent and independent class II transactivator (CIITA) pathways of MHC class II expression have been found in the eye and the brain.
While the eye may be an immune privileged tissue, tears produced by the eye's lachrymal glands do not remain in the eye. Tiny ducts, which line both eyelids and the inner edges of the eyelids, drain the constantly produced tear fluid from the surface of the eyes into the nasal cavity and throat. The nasal and oral cavities are predominantly lined with epithelial cells that show both innate and active immune responses. Both systemic and local immune responses can be generated through the immunization process. The innate response involves the local secretion of defense proteins that are uniquely expressed in the mouth, nose and upper airways. The active immune response of nasal epithelia is well known and has been successfully exploited for the delivery of vaccine antigens against a variety of infectious agents including influenza, human respiratory syncytial virus (RSV), meningococcal OpaB and OpaJ proteins, and equine herpesvirus-1 (EHV-1).
Specific peptide-induced active immunity with nasal exposure has been seen for several sequences. These responses include Th2-type T cell, cytotoxic T-cell and neutralizing antibody responses. An immunization strategy against Alzheimer's disease was examined using Abeta 1-15 or full-length Abeta 1-40/42 with the mucosal adjuvants, native labile enterotoxin (LT) or its non-toxic form, LT(R192G). Mice were immunized against the intestinal nematode Trichinella spiralis by intranasal administration of a 30-mer peptide, residues 210 to 239, from the T. spiralis 43-kDa antigen.
Immuno suppression and tolerance have also been induced with the administration of soluble protein/peptide antigens to the oral and nasal mucosa. The effects of experimental autoimmune neuritis (EAN), an animal model of the human Guillain-Barre syndrome (GBS) were attenuated after nasal administration of the neuritogenic peptide 180-199 and of the cryptic peptide 56-71 of the rat neuritogenic P0 protein of peripheral nerve myelin. Synthetic peptides corresponding to T- or CD4(+) epitopes of the acetylcholine receptor (ACHR) protein have prevented experimental myasthenia gravis (EMG) in mice.
The broad spectrum of immunological and inflammatory responses of the eye and ocular epithelium pose a significant problem in the effective treatment of ocular conditions that remain largely unresolved. Accordingly, what is needed in the art is a method of and compositions for reversibly modifying epithelial tight junctions so as to permit absorption or transport of drugs or other desirable compounds that are either presented from passage or that have decreased passing efficiency across epithelial cell layers. What is further needed is a method or composition that decreases the amount of drug or other desirable compound needed to treat conditions and/or infections of tissues protected by tight junctions. What is still further needed is a method or composition that can reversibly modify epithelial tight junctions without damaging or injuring the epithelial cells forming the tight junctions. Finally, what is further needed is methods of and compositions for decreasing transepithelial resistance in increasing ion transport across epithelial cells regulated by tight junctions.